Microarrays for Viral Pathogen Detection and...
Transcript of Microarrays for Viral Pathogen Detection and...
![Page 1: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/1.jpg)
Microarrays for Viral Pathogen Detection and Discovery
Charles Chiu, MD/PhD, Clinical Infectious Diseases FellowDepartment of Medicine, Division of Infectious Diseases
DeRisi LaboratoryUniversity of California at San Francisco
March 26, 2006
![Page 2: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/2.jpg)
Summary of Presentation
• Using the Virochip for Pathogen Detection and Discovery
• E-Predict: A Computational Tool for Virus Prediction
• Hospitalizations from Critical Illness– Case Report: a 28 year-old young adult female
with fever and hypoxia
![Page 3: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/3.jpg)
Why Pathogen Discovery?• Many common infectious diseases have unrecognized viral causes (i.e.
respiratory infections)• Many diseases once thought to be noninfectious actually have a viral
origin (i.e. cancer)
![Page 4: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/4.jpg)
• Viral culture – many viruses not culturable• Immunoassays – require special reagents, candidate
viruses• PCR / RT-PCR – limited breadth (candidate viruses)• Subtractive Hybridization – low throughput, laborious• Expression cDNA-library screening – low sensitivity
Solution: Develop a comprehensive, unbiased, and high-throughput method to detect viruses
from clinical samples
Limitations of Current Detection Methodologies
![Page 5: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/5.jpg)
• A DNA microarray allowing simultaneous screening of all known viruses
• Global approach to detection
• Develop a comprehensive picture of which viruses are present and how frequently
The Virochip
![Page 6: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/6.jpg)
The Virochip Pentathlon
WOC, 2006
![Page 7: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/7.jpg)
![Page 8: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/8.jpg)
http://derisilab.ucsf.edu/core/mGuide_v3.1.pdf
![Page 9: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/9.jpg)
Selection of Viral Sequences
![Page 10: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/10.jpg)
Example of Viral Genome Sequence Coverage (70mer oligonucleotides)
> 59 49-59 18-49
![Page 11: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/11.jpg)
![Page 12: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/12.jpg)
Virus Chip Version 322,000+ viral sequences representing all viral species in GenBank as of June 2004.
(>1.5 Megabases of sequence)
![Page 13: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/13.jpg)
“E-Predict”
Urisman,2005
![Page 14: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/14.jpg)
E-Predict: A sample from the clinic
![Page 15: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/15.jpg)
E-Predict: SARS
![Page 16: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/16.jpg)
Six Key Selection Criteria:
• defined clinical entity • epidemiological evidence for an infectious
etiology• specific target group • focal pathology (e.g. inflammation)• sample availability• clinical relevance
Which Diseases to Target with the Microarray?
![Page 17: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/17.jpg)
Current Virochip Projects
![Page 18: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/18.jpg)
Hospitalizations from Critical Illnesses
![Page 19: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/19.jpg)
The United States CDC UNEX Model
• Population-based surveillance for unexplained death and critical illness possibly due to infectious causes (UNEX) in previously healthy young individuals (Hajjeh, 2002)
• 62% of cases fatal, 79% autopsies• Infectious agent identified for only 34 / 122
cases (28%)
Goal – to use the Virochip as a comprehensive screening tool to better identify infectious agents in such cases
![Page 20: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/20.jpg)
History of present illness:• 10 day history of fever, cough, night sweats, bloody
sputum, and muscle pain• Evaluated 3 days prior and treated empirically as an
outpatient with oral azithromycin
Presentation:• Fever • Hypoxia• Elevated white blood
cell count
Case Report: a 28 year old healthy adult with fever and shortness of breath
![Page 21: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/21.jpg)
Chest CT Scan upon Admit, Representative Slice
![Page 22: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/22.jpg)
Treatment:- Patient was treated with ceftriaxone and doxycycline. - On hospital day 3, the patient progressed to acute respiratory failure- Antimicrobials were changed to moxifloxacin and oseltamivir; high-dose methylprednisolone was initiated
On hospital day 6, an open lung biopsy was performed:
Organizing bronchiolitis but no
viral inclusions, multinucleated giant
cells, or vasculitis
![Page 23: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/23.jpg)
Diagnostic Tests:• blood, urine, and sputum viral, bacterial, and fungal cultures• urine Legionella antigen• Serum rheumatoid factor and anti-nuclear antibody• Serum Cryptococcal antigen• Coccidioidomycosis, histoplasmosis, Mycoplasma, Chlamydia Ab titers• HIV serum antibody• Bordetella pertussis DFA and PCR• Immunofluorescence test for Pneumocystis jiroveci• Serology tests for blastomycosis, tularemia, sporotrichosis, Q fever, and
leptospirosis
Viral Assays:• shell vial assay for cytomegalovirus• DFA tests for respiratory syncytial virus, adenovirus, influenza A/B,
parainfluenza virus types 1, 2, and 3• Metapneumovirus PCR• SARS coronavirus PCR• ELISA for hantavirus (Sin Nombre)
All tests returned negative! ViroChip Analysis:Endotracheal aspirate from hospital day 8.
![Page 24: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/24.jpg)
ViroChip Result:Parainfluenza-4 (HPIV-4)
Confirmation by specific PCR: NEJM
Human Parainfluenza Type 4
![Page 25: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/25.jpg)
ViroChip Result:Parainfluenza-4 (HPIV-4)
Confirmation by serology:
IFA with hospital day 21 sera (1:512)
Human Parainfluenza Type 4
![Page 26: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/26.jpg)
Parainfluenza Virus
(Linda Stannard, University of Cape Town, S.A.)Hall CB. NEJM. 2001;344:1917-28.
• ssRNA virus• 4 serotypes
– Type 1: Frequent cause of croup(laryngotracheo-bronchitis) in children
– Type 2: Similar to Type 1 but less severe disease
– Type 3: Important cause of bronchiolitis and pneumonia
– Type 4: Generally mild disease
![Page 27: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/27.jpg)
Virus DFA Kits
Standard kits do NOT screen for
PIV-4!
![Page 28: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/28.jpg)
Acknowledgements
DeRisi Virochip GroupJoseph DeRisi Don Ganem
Anatoly UrismanAmy KistlerKael FischerNicole FischerPatrick TangSilvi Reich
Hospitalization from Critical IllnessBruce Patterson, StanfordJason Derek Merker, Stanford
![Page 29: Microarrays for Viral Pathogen Detection and Discoverysites.nationalacademies.org/cs/groups/pgasite/documents/webpage/... · Microarrays for Viral Pathogen Detection and Discovery](https://reader031.fdocuments.net/reader031/viewer/2022022805/5caee25388c9938f4d8dc7b3/html5/thumbnails/29.jpg)
Viral Discovery Follow-Up
Phase I – Short-term:• Clone the genome from multiple samples / patients• Recover or generate infectious clones• Generate PCR-based diagnostic for rapid screening• Develop probes for in situ hybridization assays• Acquire more samples from another source
Phase II – Long-term (linking the virus to the disease):• Develop immunoassay for rapid tests• Expand screening to larger population• Cell culture system for viral study• Characterize individual proteins• Establish an animal model